Project Summary Breathing patterns have strong impacts on emotions in humans. Voluntary control of respiration, especially via nasal breathing as practiced in yoga and meditation, is effective in reducing anxiety, stress, or even panic attacks. The effects of breathing patterns on emotions are thought to be related to respiration-entrained brain rhythms, which have been recognized for decades, but their sources and functions remain elusive. One potential source of respiration-entrained brain activity is the olfactory system. Nasal airflow activates intrinsically mechanosensitive olfactory sensory neurons (OSNs) in the nose, which carry the information to the olfactory bulb (OB) and subsequently to the olfactory cortical regions including the anterior olfactory nucleus/taenia tecta (AON for simplicity). It is well known that the neural activity along the olfactory pathway is highly correlated with respiration. Interestingly, recent studies indicate that many non-olfactory cortical and limbic structures including the medial prefrontal cortex (mPFC) also display nasal airflow-dependent, respiration-entrained oscillations in both rodents and humans. A potential role of respiration-entrained neural activity has been examined in the context of learned fear, an emotional state inferred by quantifiable freezing behavior in rodents. During fear retrieval after tone-foot shock pairing, mice freeze to the conditioned tones while breathe at a steady rate (~4 Hz), which is correlated with a predominant 4-Hz oscillation in the mPFC, a region critical for expression of conditioned fear. Disruption of peripheral olfactory inputs significantly reduces the 4-Hz oscillation in the mPFC and leads to prolonged freezing periods. However, the neural circuits underlying the effects of olfactory inputs on the mPFC activity and fear-related behaviors remain largely unresolved. We recently discovered that the mPFC receives direct inputs from the AON, a major target of the OB tufted cells, which receive stronger peripheral inputs and display robust respiration-entrained activity. The central hypothesis of this proposal is that the OB tufted cells?AON?mPFC pathway is the critical neural circuit in modulating the mPFC respiration-related rhythm and relevant behaviors. Multidisciplinary approaches (gene editing, ex vivo and in vivo electrophysiology, optogenetics, circuit tracing, and behavior) will be combined to pursue three specific aims. We will (1) dissect out this neural pathway in a cell-type specific manner, (2) determine functional properties of this pathway, and (3) determine behavioral effects of optogenetic manipulations of this pathway. Overall, the current study will provide critical insights into olfactory modulation of respiration-entrained brain activity and behavior.